It has long been understood that morbidity and mortality caused by cancer, especially in view of the fact that surgical techniques are readily available, are due to more-or-less systemic sequelae of the original multiplication of the cancer cells. Metastasis of an original tumor to additional locations, the destruction of target locations as a direct or indirect result of this metastasis, cachexia, hypercalcemia, and other symptomologies characterize the course of the malignancy. The mechanisms whereby these sequelae occur are believed to involve a variety of cytokines, growth factors, and cell adhesion molecules, among other factors. It is entirely unknown, however, how cancers dissolve bone in bone metastases since at present, the only cells known to be able to dissolve bone are osteoclasts. It is not known how metastasized cancer dissolves bone in order to accommodate the growth of the cancer.
One of many factors which is known to play a role in at least some of these processes is parathyroid hormone-related protein (PTH-rp). Due to its similarity to PTH, it has been recognized as a mediator of humoral hypercalcemia of malignancy (HHM). In this role, PTH-rp secreted by the tumor is circulated through the blood and is associated with hypercalcemia, an index of bone resorption. PTH-rp has been shown to be the cause of systemic bone resorption and calcium reabsorption from urine at the tubule. Both of these result in a systemic elevation of blood calcium levels. This factor has been described in connection with HHM in U.S. Pat. No. 5,116,952. This patent also describes preparation of anti-PTH-rp antibodies using peptide subunits of the partially sequenced protein. This protein has also been called adenylate cyclase stimulating factor (ACSF) in U.S. Pat. No. 5,312,810 and humoral hypercalcemic factor (hHCF) in U.S. Pat. No. 5,114,843. The association of PTH-rp with HHM is also described by Yates, A. J. P. et al., J Clin Invest (1988) 81:932-938. Japanese application A4-228089, published Aug. 18, 1992 describes recombinant production of anti-PTH-rp antibodies, including murine/human chimeras. This application also discloses animal studies demonstrating that either murine or chimeric monoclonal antibodies directed to PTH-rp ameliorate the effects of HHM in a model system where HHM is caused by either systemically infusing PTH-rp into mice peritoneally or by implanting PTH-rp-producing prostate cancer cell lines in nude mice.
PTH-rp has been purified from human lung cancer, breast cancer and renal cell carcinoma. Although PTH-rp was originally found associated with tumor cells, it is also present widely in normal tissue. The gene encoding PTH-rp was cloned and expressed by Suva, L. J. et al. Science (1987) 237:893-895. PTH-rp has been shown to bind to PTH receptors (Abou-Samra, A. et al. Proc Natl Acad Sci USA (1992) 89:2732-2736) and has activities similar to PTH (Horiuchi, N. et al. Science (1987) 23:1566-1568). It has been shown to stimulate adenylate cyclase in renal and bone systems, increase tubular reabsorption of calcium, decrease renal phosphate uptake and stimulate 1.alpha.-hydroxylase. Thus, it is logical that where PTH-rp is circulating, inhibitors of the PTH-like action of PTH-rp would interfere with these biological events, including calcium reabsorption from urine to blood at the kidney tubules and systemic bone resorption. However, PTH/PTH-rp receptors are not found on osteoclasts. Either such receptors have been resistant to characterization or to the extent that PTH-rp may be found to affect osteoclasts, the effect may be exerted by some unknown mechanism, for example by effecting the secretion of unknown mediators from osteoblasts (which are known to have PTH/PTH-rp receptors). There is no available evidence to connect PTH-rp to bone resorption mediated by osteoclasts.
PTH-rp also has properties that are not shared by PTH, including regulation of placental calcium transport as summarized in a review by Mallette, L. E. Endocrine Rev (1991) 12:110-117. Of particular relevance with respect to the invention herein is that PTH-rp has also been associated with the establishment of bone metastasis in breast cancer (Powell, G. J. Cancer Res (1991) 51:3059-3061; Southby, J. et al. Cancer Res (1990) 50:7710-7716). The data provided in these disclosures show only the association of PTH-rp with bone metastasis of breast cancer, but no causal relationships are implied. PTH-rp has been considered as a possible autocrine factor for a limited number of tumors (Burton, P. B. J. et al. Biochem Biophys Res Com (1990) 167:1134-1138; Li, X. et al. Cancer Res (1993) 53:2980-2986).
Thus, the precise role of PTH-rp in mediating the effects of a primary cancer is not well understood. Not only are there additional factors which also participate in the progress of this condition, the production of PTH-rp is believed affected by additional factors such as prolactin, glucocorticoids, epidermal growth factor, TGF-.alpha., TGF-.beta., estrogen, "stretch," and even extracellular calcium concentration. The presence of additional factors is particularly important and adds to the uncertainty of the mechanism. Factors such as IL-1, IL-6, IL-11, M-CSF and GM-CSF in particular have been considered important in bone resorption. In a report by Sasaki, A. et al. J Bone Min Res (1994) 9 Supp 1:S 294, B 257, it is demonstrated that administration of recombinant human IL-1.alpha. into calvaria of nude mice attracted metastasis of cancer calls that had been inoculated into the left ventricle, while other bones of the same animal which did not receive IL-1.alpha. did not show cancer metastasis. Gilles, J. et al. ibid. S 337, B 433, suggest that IL-6 markedly potentiates the effects of bone resorbing factors on osteoclastic bone resorption and that IL-6 production in bone is important both in cytokine-mediated bone resorption and in those disease states where cytokine-mediated bone resorption is potentially involved, such as estrogen-related bone loss, myeloma and Paget's Disease. It has also been shown that transgenic mice that are modified to comprise an IL-6 gene knockout show reduced bone loss in models for osteoporosis. It has been confirmed that osteoclasts have receptors for IL-6 and IL-11. Osteoclasts treated with M-CSF and GM-CSF show accelerated maturation in culture. Transgenic mice that have genetic deficiencies in the M-CSF gene also exhibit a condition mimicking osteoporosis and this condition can be treated by injection of M-CSF. Thus, a multiplicity of factors are known which appear to play a major role in local bone resorption and/or osteoclast activation.
Sato, K. et al. J Bone Min Res (1993) 8:849-860 describe results obtained in a murine model of HHM wherein the affected mice were administered a monoclonal murine antibody obtained from immortalized spleen cells of mice injected with a peptide representing amino acid positions 1-34 of PTH-rp. Passive immunization resulted in decreases in serum calcium concentration in these hypercalcemic nude mice that had been transplanted with human PTH-rp-producing tumors. These tumors were implanted in nonbone tissue to provide a model for HHM. The authors suggest that if a human counterpart to this antibody could be obtained, it might be used in treatment where malignancy-associated hypercalcemia is due to PTH-rp. (Sato, K. et al. use the term "malignancy-associated hypercalcemia" in this paper; it is synonymous with HHM.) In this model, elevated levels of PTH-rp are maintained in the blood which are, evidently, mitigated by the administration of anti-PTH-rp antibodies. Similar studies were conducted by Kukreja, S. C. J Clin Invest (1988) 82:1798-1802 and showed that polyclonal anti-PTH-rp antibodies suppress hypercalcemia in a human tumor model of HHM. This is the first disclosure of any therapeutic effect of anti-PTH-rp on HHM. The relevance of this model is verified by the finding of Tashjian, A. H. et al. J Exp Med (1964) 119:467 that 15% of 147 hypercalcemic breast cancer patients exhibited no bone metastases.
Hypercalcemia may also be caused by osteolysis of bone through the mediation of osteoclasts. Mundy, G. R. J Clin Invest (1988) 82:1-6 describes increased osteoclastic bone resorption in areas surrounding breast cancer metastases. In addition, breast cancer cells have been shown to resorb bone directly in vitro (Eilon, G. & Mundy G. R., Nature (1978) 276:726-728.
Kohno, N. et al. Surgery Today, Japan J Surg (1993) 4:215-220 reported studies performed on immobilized sections of surgically removed breast cancers using an anti-PTH-rp antibody also prepared by immunizing mice with the first 34 amino acids of PTH-rp. The antibody bound to 57% of the tumors; it bound to 83% of the tumors derived from patients who developed skeletal metastases but only 38% in those who either developed lung metastases or no metastases at all. These authors conclude that their results suggest that PTH-rp-positive tumors have an affinity to bone. However, the observations related only to the staining of cancer tissues by anti-PTH-rp and are not quantitative. Thus, the results do not show a causal relationship between the presence of PTH-rp and any acceleration of bone metastasis or growth enhancers. An equally plausible explanation is that PTH-rp is an effect rather than a cause of metastasis, cancer growth and/or osteolysis.
The present inventors have developed a model of human breast cell cancer metastasis to bone that results in osteolysis. Nakai, M. et al. Cancer Res (1992) 52:5395-5399; Sasakai, A. et al. J Bone Min Res (1993) 8 (Supp 1):No. 92. Tumor cells introduced into the left cardiac ventricle of nude mice can be shown to cause osteolytic lesions that can be seen by x-ray examination and that can be confirmed histologically. A375 melanoma cells and the human breast cancer cell line MDA231 have been used in this model. Using this model, it was demonstrated that bone metastasis could be prevented by a synthetic antagonist to laminin.
This model has also been used to demonstrate in vivo the metastatic nature of human neuroblastoma cells as described in an abstract by Yoneda, T. et al. J Bone Min Res (1994) 9 (Supp 1):S 293, B 225. Six weeks after inoculation with the neuroblastoma cells, x-rays showed lesions in the tibiae that were both osteolytic and osteoblastic, but there was no hypercalcemia or cachexia. Thus, cancer metastasis of the bone is clearly a complex situation which can generate both osteoblasts and osteoclasts and is not always associated with systemic disorders such as hypercalcemia or cachexia. Histologic examination showed that neuroblastoma cells occupied the bone marrow cavity, and there were numerous bone resorbing osteoclasts (which have not been demonstrated to exhibit PTH/PTH-rp receptors) on endosteal bone surfaces. Culture supernatants of the tumor cells stimulated bone resorption in organ cultures of fetal rat long bones and increased proliferation of osteoblastic osteosarcoma cells, which promote bone formation. The culture media contained significant levels of PTH-rp and high levels of IL-6. It is known that osteoclasts have IL-6 receptors.
The present inventors have also demonstrated that the number of osteolytic lesions is increased by enhancing PTH-rp expression of the MDA231 cells used in the model, but not diminished by MDA231 cells transfected with an antisense PTH-rp construct. Overexpressing clones were obtained by transfecting MDA231 cells with cDNA encoding human prepro PTH-rp; clones having diminished production of this peptide were obtained by transfecting the cell line with an antisense construct. A clone showing elevated PTH-rp expression (100 pM/24 hours) produced 16.3.+-.3.8 lesions radiographically at 3 weeks as compared with the antisense construct which secreted less than 0.3 pM/24 hours which produced only 7.6.+-.0.22 lesions. The number of lesions in mice administered unmodified MDA231 cells which produce 1.6-8.4 pM/24 hours PTH-rp is similar to the number of lesions in mice administered the MDA231 cells modified to contain the antisense construct, which produces less PTH-rp. No plasma concentrations of PTH-rp were detectable in any of the mice used in these studies; hypercalcemia was minimal and present only in mice harboring the PTH-rp-enhanced MDA231 cells. The present inventors have also shown that PTH-rp concentrations in serum-free media conditioned by MDA231 cells were increased two-fold when the cells were cultured on an extracellular matrix produced by bone cells. These results were reported by Guise, T. A. et al. J Bone Min Res (1994) 9 (Supp 1):S 128, No. 30.
It has now been found that antibodies specific for PTH-rp are affective in inhibiting metastasis and ameliorating the effects of malignant cells localized in bone. This provides an effective pharmacological approach to treatment.